A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase

Shuhui Niu; Fang Liu; Yaping Wang; Ben Rao; Yueying Wang

doi:10.3390/molecules29081861

A Study on the Efficient Preparation of α-Ketoglutarate with L-Glutamate Oxidase

Molecules. 2024 Apr 19;29(8):1861. doi: 10.3390/molecules29081861.

Authors

Shuhui Niu^{1

2}, Fang Liu¹, Yaping Wang², Ben Rao¹, Yueying Wang¹

Affiliations

¹ National Biopesticide Engineering Technology Research Center, Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Sciences, Biopesticide Branch of Hubei Innovation Centre of Agricultural Science and Technology, Wuhan 430064, China.
² State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty, Hubei University, Wuhan 430062, China.

Abstract

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOX^Str) from Streptomyces viridosporus R111 and catalase (KatG^Esc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOX^Str r) and catalase (KatG^Esc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOX^Str and KatG^Esc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

Keywords: L-glutamate oxidase; alpha-ketoglutaric acid (α-KG); catalase; whole-cell catalysis.

MeSH terms

Amino Acid Oxidoreductases / chemistry
Amino Acid Oxidoreductases / metabolism
Catalase* / chemistry
Catalase* / metabolism
Enzymes, Immobilized / chemistry
Enzymes, Immobilized / metabolism
Escherichia coli* / enzymology
Ketoglutaric Acids* / chemistry
Ketoglutaric Acids* / metabolism
Streptomyces / enzymology

Substances

Ketoglutaric Acids
L-glutamate oxidase
Catalase
Amino Acid Oxidoreductases
Enzymes, Immobilized

Grants and funding

This research was funded by the National Key R&D Program of China (2023YFD1201605-1), Provincial-Level Science and Technology Innovation Special Fund (Science and Technology Talent Service Enterprise Project: 2023DJC123), the Wuhan East Lake New Technology Development Zone “Unveiling the List” project (2022KJB001), Hubei Provincial Natural Science Foundation Key Project (Outstanding Youth, 2021CFA063), Wuhan Science and Technology Major Project (2023020302020708), and Wuhan’s major science and technology project for “Bottleneck” technology in public relations (2023020302020708).